|Name of the Death Receptor|
(Synonyms in brackets)
|Ligand(s) of the Death Receptor|
(Synonyms in brackets)
|Fas (CD95, Apo1)||CD95L (FasL) and DAXX|
|TNFR1 (p55, CD120a)||TNF and Lymphotoxin alpha|
|DR3 (Apo3, WSL-1, TRAMP, LARD)||Apo3L (TWEAK)|
|DR5 (Apo2, TRAIL-R2, TRICK2, KILLER)||TRAIL (Apo2L)|
All those Death Receptors belong to the TNFR family. Other TNFR family members, which are no death receptors, include the low affinity NGF receptor, B cell antigen CD40, T cell antigens OX40, CD27, 4-1BB, and Hodgkins's Lymphoma antigen CD30.
The Fas gene (12 kb, 9 exons, located on human chromosome 10q) is expressed in different cell lines and tissues with considerable variability. In mouse, many tissues and cell lines only weakly express Fas, but abundant expression was found in mouse thymus, liver, heart, lung, kidney and ovary. Unlike mouse thymocytes human thymocytes only weakly express Fas. In mouse thymocytes, Fas is expressed in almost all populations except for double-negative (CD4-CD8-) thymocytes.
Fas is highly expressed in activated mature thymocytes or lymphocytes transformed with HTLV-1, HIV and EPB virus. Some other tumor cells also express Fas, although the expression level is low in comparison to lymphoblastoid cells. The expression is up-regulated by interferon-gamma in various cell lines, or by a combination of IFN-gamma and TNF-alpha in human B cells which may explain the enhancement of the cytotoxic activity of anti-Fas by these cytokines.
Fas Ligand (FasL)
FasL was purified in 1993 from the solubilized membrane fraction of a cytotoxic T cell line that was observed to kill target cells expressing Fas but not cells which did not display Fas. Finally FasL cDNA was cloned (Takahashi et al., 1994, Cell, 76: 969); the Fas gene is located on human chromosome 1 and comprises 5 exons. The Fas gene encodes a 40 kDa protein which has no signal sequence at the N-terminus, but a domain of hydrophobic aa in the middle of the molecule, indicating that it is a type II membrane protein with the C-terminal region outside the cell. This extracellular region has significant homology to the corresponding region of other members of the TNF family. FasL is expressed almost exclusively by activated T cell lines. Expression of FasL can be induced in T cells through activation of the T cell receptor.
The Fas Pathway
FasL is a homotrimeric membrane-molecule; each FasL trimer binds three Fas receptor molecules on the surface of the target cell. This results in the clustering of the receptors' death domains (DDs) which then recruit the cytosolic adapter protein FADD by binding to FADD's death domains. FADD not only contains a DD but also a Death Effector Domain (DED) that binds to an analogous domain repeated in tandem within the zymogen form of Caspase-8. The complex of Fas receptor (trimer), FADD and Caspase-8 is called the Death Inducing Signaling Complex (DISC). Upon recruitment by FADD, caspase-8 oligomerization drives its activation through self-cleavage (Muzio et al., 1998, J. Biol. Chem., 273: 2926). Active Caspase-8 then activates downstream caspases, committing the cell to apoptosis.
In Fas-mediated apoptosis several signaling pathways may be involved in the activation of the executing death machinery:
Type I and type II Fas-sensitive cell lines
Recently, two types of cell lines were defined which differ in their kinetics of caspase activation (Scaffidi et al., 1998, EMB, 17(6): 1675-87). Upon Fas-ligation, type I cells (e.g. B lymphoblastoid cell line SKW6.4 and T lymphoma cell line H9) show early Caspase-8 and Caspase-3 activation, while type II cells (e.g. Jurkat and CEM) show delayed Caspase-8 and -3 activation. Nevertheless, both types of cells have similar kinetics of apoptosis. The two cell types also do not differ with respect to their actiation of mitochondria during Fas-mediated apoptosis: in all cases disruption of the mitochondrial membrane potential (deltapsi) was detected with similar kinetics, and cytochrome c was released from the mitochondria. Differences were seen in the effect of Bcl-2 on Fas-mediated apoptosis: upon Bcl-2 overexpression type I cells were not affected in their Fas-sensitivity, whereas Bcl-2 transfected type II cells (Jurkat) were significantly resistant to apoptosis after Fas-triggering. Interestingly, Bcl-2 inhibited deltaPSI loss and cytochrome c release from mitochondria in type II cells as well as in type I cells! This suggests that the mitochondrial pathway is intact and activated in both type of cells, but only type II cells depend on the mitochondrial pathway while type I cells additionally possess an alternative pathway. Indeed, Bcl-2 overexpression blocked the activation of Caspase-8 and Caspase-3 in type II cell line Jurkat, whereas type I cells were not inhibited in the activation of Caspase-8 and Caspase-3. These observations suggest that in type II cells strong activation of caspase-8 and -3 occurs at a level downstream of mitochondria and depends on mitochondrial release of cytochrome c, that can be blocked by Bcl-2. In contrast, in type I cells Caspase-8 may be activated directly at the DISC level, and Fas-mediated apoptosis is independent of the mitochondrial pathway and therefore independent of Bcl-2. Indeed, immunoprecipitation experiments demonstrated that upon Fas-ligation DISC formation (recruitment of FADD and Caspase-8 to the receptor complex) is strongly reduced in Type II cell lines Jurkat and CEM, while in type I cell DISC formation was strong.
It was proposed that the reduced FADD and Caspase-8 recruitment to the DISC in type II cells could be caused by a protein bound to Fas, blocking DISC formation. A 120 kDa candidate protein was identified that was only found in the immunoprecipitated DISC from type II cells and that may block DISC formation (Medema et al, 1997, EMBO, 16: 2794-2804).
Fas-mediated apoptosis and Ceramide Pathway
Ceramides are known stimuli of apoptosis and are released by activation of an acidic and/or neutral sphingomyelinase. Both enzymes have been shown to be activated by the Fas receptor (Cifone et al., 1995, EMBO, 14: 5859-5868; Tepper Et al., 1995, Proc. Nat. Acad. Sci. USA, 92: 8443-8447). It also was reported that Fas-mediated apoptosis can be partially inhibited by direct inhibition of acidic sphingomyelinase using the drug imipramine. Treatment of Jurkat cells with caspase inhibitors (CrmA, Ac-YVAD-cmk, zVAD) not only inhibited Fas-mediated apoptosis but also resulted in a decrease of acid sphingomyelinase activity and consequently in inhibition of sphingomyelin consumption and ceramide release. Caspase inhibitors also were demonstrated to prevent activation of JNK and p38-K, which normally are activated tenfold upon Fas-ligation in Jurkat. It was proposed that upon Fas-triggering early caspases (Caspase-8/ maybe Caspase-1) stimulate directly or indirectly the acidic sphinomyelinase resulting in the release of ceramide (Brenner et al., 1998, Cell death and Diff., 5: 29-37).
A family of viral proteins called vFLIPs and a related cellular protein called cFLIP contain a DED that is similar to the corresponding segment in FADD and Caspase-8. The role of FLIP is controversial, as FLIP overexpressionn either inhibits or activates apoptosis (Wallach, 1997, Nature, 388: 123).
Besides FADD, DAXX is another Fas-binding protein (Yang et al., 1997, Cell, 89: 1067-1076). DAXX (Death-Domain Associated protein) binds to the Fas death domain although DAXX itself does not contain a DD. It also interacts with the DD of TNFR1 but not with the intracellular region of CD40, a closely related receptor that lacks a death domain.
Inhibitor studies suggest that Daxx and FADD bind independently to Fas and activate distinct pathways. DAXX can enhance Fas-mediated apoptosis by activating the JNK kinase cascade, culminating in the phosphorylation and activation of transcription factors such as c-Jun. It was reported that ASK1 (Apoptosis Signal Regulating Kinase 1) interacts directly with DAXX and, indeed, is activated by DAXX (Chang et al., 1998, Science, 281: 1860-1863). ASK1 is a MAP kinase kinase kinase (MAP3k) that upon TNF-alpha stimulation mediates apoptosis by activation of the JNK and p38 MAP kinase cascades (Ichijo et al., 1997, Science, 275, 90-94). JNK is activated by apoptotic stimuli such as anti-Fas, TNF-alpha, UV and transforming H-Ras. Upon DAXX overexpression, the activation of JNK is not inhibited by caspase inhibitors, but subsequent apoptosis is blocked. This would suggest that apoptotic JNK activation is located upstream of the caspase-cascade. The JNK pathway culminates in the activation of transcription factors which directly or indirectly may counteract the expression of survival factors, such as NFkB or Bcl-2. The JNK pathway itself can be blocked by Bcl-2. It is not yet clear which impact the DAXX-activated JNK pathway has in Fas-mediated apoptosis; the requirement for the JNK pathway in Fas-mediated apoptosis seems to be cell-type specific.
Physiological Roles of Fas/FasL
Fas/FasL and Negative Selection of Lymphocytes
Studies on mice with spontaneous mutations in the Fas gene (lpr mutations) or mutations in the FasL gene (gld mutations) showed an abnormal accumulation of lymphocytes in those mice. This suggests that Fas/FasL are involved in normal lymphocyte death.
In the life of lymphocytes, both T cells and B cells normally die at various stages of their development. Precursor T cells originate in the bone marrow and migrate to the thymus where they mature into single positive (CD4+CD8- or CD4-CD8+) T lymphocytes. The T cells that can interact with self-MHC expressed in the thymus are positively selected (positive selection) while those that cannot interact with self-MHC die by apoptosis. T cells that strongly react with self antigen presented by self-MHC also are induced to undergo apoptosis (negative selection). More than 95% of the T cells that migrate into the thymus die there; the remaining 5% migrate to the peripheral lymphoid organs as mature T lymphocytes. In the periphery, the mature T cells again undergo an additional selection process. Those that interacted with the self antigens expressed only in the peripheral tissues would die (peripheral clonal deletion). Furthermore there must be some mechanism in the periphery to eliminate lymphocytes after they have been activated by antigen to ensure that the organism does not fill up with activated lymphocytes.
Positive and negative selection in the thymus are apparently normal in lpr mice, but peripheral clonal deletion and the elimination of activated T cells are impaired in lpr and gld mice: in lpr and gld mice, an antigen can stimulate the proliferation of mature T cells, but the subsequent death process is severely retarded, both in vitro and in vivo. So, the Fas system is normally involved in both the clonal deletion of autoreactive T cells in peripheral lymphoid organs and the elimination of activated T cells (AICD) after they have responded to foreign antigens.
B cells are also thought to die by apoptosis at several steps of their development. During development in the bone marrow, the B cells that are strongly self-reactive to self-components are deleted, apparently by a Fas-independent mechanism. The surviving B cells then migrate to peripheral lymphoid organs where thy can be activated by antigen. Activation of mature B cells causes the expression of Fas and renders the cells sensitive to fas-mediated killing by anti-Fas. It is possible that FasL on activated T lymphocytes binds to fas on activated autoaggressive B cells and kills them by apoptosis (Nagata and Goldstein, 1995, Science, 267: 1449).
Fas in T cell-mediated Cytotoxicity
Cytotoxic T lymphocytes (CTLs) are the main effectors of the immune system for the elimination of virus-infected cells. There are two main pathways of T cell-mediated cytotoxicity: one of them is the well-known perforin-granzyme-based mechanism (Kägi et al., 1994, Nature, 369: 31), which is Ca2+-dependent. In the presence of EGTA-Mg2+ this perforin-granzyme-based mechanism can be suppressed while the Ca2+- independent Fas-based cytotoxic mechanism remains active. During the Fas-based cytotoxic response, the cytotoxic cell produces FasL upon recognition of the target cell. Then, FasL on the cytotoxic cell crosslinks the Fas receptor on the target cell what induces the intrinsic suicide program of the target cell. While CD4+ helper T cells express FasL and possess Fas-based cytotoxicity (and usually possess no perforin-granzyme activity), professional cytotoxic CD8+ T cells usually express both, the Fas-based and the perforin-based mechanism. There is not much cross-talk between those two pathways, since cytotoxic cells from perforin knockout mice still can lyse target cells by means of the Fas pathway, whereas cytotoxic cells bearing gld mutations still are cytotoxic due to the perforin-granzyme pathway.
Fas and FasL in Pathology
One category of Fas-related diseases results from a defective Fas/FasL system such as found in gld and lpr mutations in mice. Accumulation of peripheral lymphoid cells (including autoimmune cells) and formation of autoreactive antibodies can result in lymphadenopathy, splenomegaly, nephritis and arthritis. In some patients elevated levels of a soluble form of Fas protein in the blood serum is supposed to be responsible for the observed SLE phenotype.
Another category of Fas-related diseases may be caused by excessive activity of the Fas system. For example, crosslinking of the CD4-receptor increases the expression of Fas in peripheral PBLs (Peripheral Blood Lymphocytes). Indeed, human T cell lines transformed with HIV are more sensitive to Fas-mediated apoptosis than non-infected cells and HIV-infected children abundantly express Fas on T-lymphocytes: Fas might be involved in the pathology of AIDS (Debatin et al., 1994, Blood, 83: 3101). On the other hand, tumors that constitutively express FasL might suppress immune surveillance by eliminating tumor-reactive immune cells (Strand et al., 1996, Nature Med., 2: 1361).